19-norpregnane steroids as neurochemical initiators of change in human hypothalamic function

ABSTRACT

Several 19-nor-pregnanes are provided which have the capacity to neurochemically alter the hypothalamic function in an individual through nasal administration. These pharmaceutically active compounds can be administered by themselves or in the form of a pharmaceutical composition containing one or more pharmaceutically acceptable carriers to produce the desired effect.

This application is a continuation of, and claims the benefit under 35USC 120 of, App. Ser. No. 08/485,615, file Jun. 7, 1995, now U.S. Pat.No. 5,792,757, which is a continuation-in-part of, and claims thebenefit under 35 USC 120 of, App. Ser. No. 08/286,073, filed Aug. 4,1994, now U.S. Pat. No. 5,563,131.

CROSS REFERENCE TO RELATED APPLICATIONS

The subject matter of this application is related to the subject matterof U.S. application Ser. No. 08/127,908, filed Sep. 28, 1993 nowabandoned which is a continuation-in-part of U.S. application Ser. No.07/903,604, filed Jun. 24, 1992 now abandoned, which in turn is acontinuation-in-part of U.S. application Ser. No. 07/708,936, filed May31, 1991 now abandoned, which in turn is a continuation-in-part of U.S.application Ser. No. 07/638,185, filed Jan. 7, 1991 , now abandoned.

The subject matter of this application also relates to the subjectmatter of U.S. application Ser. No. 08/127,980 filed Sep. 28, 1993 nowU.S. Pat. No. 5,783,571 issued Jul. 21, 1998 which is anothercontinuation-in-part of U.S. patent application Ser. No. 07/903,604 nowabandoned, U.S. patent application Ser. No. 08/077,359, filed Jun. 15,1993 now abandoned, and to commonly assigned, co-pending U.S. patentapplication Ser. No. 07/903,525, filed Jun. 24, 1992 now abandoned (acontinuation-in-part of U.S. application Ser. No. 07/707,862, filed May31, 1991 now abandoned, which in turn is a continuation in-part of U.S.application Ser. No. 07/638,743, filed Jan. 7, 1991, now abandoned)entitled “Estrene Steroids as Neurochemical Initiators of Change inHuman Hypothalamic Function and Related Pharmaceutical Compositions andMethods”; and to the commonly assigned, co-pending continuation-in-partof Ser. No. 07/903,525 now abandoned, U.S. patent application Ser. No.08/077,140 now abandoned. The aforementioned U.S. patent applicationsare each incorporated herein by reference.

Finally, the subject matter of this application may relate to thesubject matter of U.S. patent application entitled “FragranceCompositions Containing Human Pheromones now issued as U.S. Pat. No.5,278,141 on Jan. 11th, 1994, which is a continuation of an applicationof the same name, filed Mar. 24, 1992, U.S. Ser. No. 07/856,435 nowabandoned.

TECHNICAL FIELD

This invention relates generally to pharmaceutical compositions andmethods for effectuating change in human hypothalamic function, therebyaltering certain behavior and physiology mediated by the hypothalamus ofindividuals. More particularly, the invention relates to the use of19-nor-pregnane steroids as neurochemical effectuators of physiology andbehavior.

DESCRIPTION OF THE RELATED ART

The present invention relates to certain compounds, namely19-norpregnane steroids, particularly 19-norpregnane steroids andrelated compounds as will be described herein, and methods of usingthese compounds as human vomeropherins in order to alter hypothalamicfunction, thereby affecting certain consequent behavior and physiology,e.g., the reduction of anxiety. The 19-nor-pregnane steroids arecharacterized by a four ring steroidal structure, methylation at the 13position and ethylation at the 17-position. The 19-nor-pregnenes are asubset which have at least one double bond.

Ohloff, G. et al. (Helv. Chim. Acta (1983) 66:192-217), which isincorporated herein by reference, have shown that several steroids(androstenes) have an odor which varies with different isomeric,diastereomeric, and enantiomeric forms. Some members of this group havebeen reported to act as a pheromone in some mammalian species forinstance, 5α-androst-16-en-3-one and 5α-androst-16en-3α-ol in pigs(Melrose, D. R., et al., Br. vet. J. (1971) 127:497-502). These16-androstenes produced by the boar induce mating behavior in estrussows (Claus, et al., Experimentia (1979) 35:1674-1675).

In some studies it has been noted that, in some species, variouscharacteristics of certain 16-androstenes (including5α-Androst-16-en-3α-ol and 5 a -Androst-16-en-3-one), such asconcentration, metabolism, and localization, are sexually dimorphic(Brooksbank et al., J. Endocr. (1972) 52:239-251; Claus, et al., J.Endocr. (1976) 68:483-484; Rwan, et al., Med. Sci. Res. (1987)15:1443-1444). For instance, 5α-Androst-16-en-3α-ol and5α-Androst-16-en-3-one, as well as Androsta-4,16-dien-3-one, have beenfound at different concentrations in the peripheral blood, saliva andaxillary secretions of men and of women (Kwan, T. X., et al., Med. Sci.Res. (1987) 15:1443-1444), and their function as a human pheromone, tothe extent of affecting choice and judgement, has been suggested (Id.;see also Gower, et al., “The Significance of Odorous Steroids inAxillary Odour”, In, Perfumery, pp. 68-72, Van Toller and Dodd, Eds.,Chapman and Hall, 1988); Kirk-Smith, D. A., et al., Res. Comm. Psychol.Psychiat. Behav. (1978) 3:379). Androstenol (5α-androst-16-en-3α-ol) hasbeen claimed to exhibit a pheromone-like activity in a commercial men'scologne and women's perfume (Andron for men and Andron for women byJovan). Japanese Kokai No. 2295916, refers to perfume compositionscontaining androstenol and/or its analogues. 5α-Androstadien-3β-ol (andperhaps the 3α-ol) has also been identified in human axillary secretion(Gower, et al., Supra at 57-60. On the other hand, there is littleagreement in the literature as to whether or not any putative pheromoneactually plays any role in the sexual or reproductive behavior ofmammals, particularly of humans. See: Beauchamp, G. X., et al., “ThePheromone Concept in Mammalian Chemical Communication: A Critique”, In:Mammalian Olfaction. Reproductive Processes and Behavior, Doty R. L.,Ed., Academic Press, 1976). See also: Gower, et al., supra at 68-73.

The pheromone properties of some estrene steroids for some mammalianspecies have been described. Michael, R. P. et al., Nature (1968)218:746 refers to Estrogens (particularly Estradiol) as a pheromonalattractant of male rhesus monkeys. Parrot, R. F., Hormones and Behavior(1976) 7:207-21S, reports Estradiol benzoate injection induces matingbehavior in ovariectomized rats; and the role of the blood level ofEstradiol in make sexual response (Phoenix, C. H., Physiol. and Behavior(1976) 16:305-310) and female sexual response (Phoenix, C. H., Hormonesand Behavior (1977) 8:356-362) in Rhesus monkeys has been described. Onthe other hand, there is little agreement in the literature as towhether or not pheromones as such play any role in the reproductivebehavior and interpersonal communication of mammals (Beuchamp, G. K., etal., “The Pheromone Concept in Mammalian Chemical Communication: ACritique”, In: Mammalian Olfaction Reproductive Processes. and Behavior,Doty, R. L., Ed., Academic Press, 1976).

An embodiment of the subject invention concerns the non-systemic, nasaladministration of certain 19-nor-pregnane and 19-nor-pregnene steroidsto affect a specific behavioral or physiological response in humansubjects, e.g., a reduction of negative affect, mood, and charactertraits. In particular, nasal administration provides for contactingneurochemical receptors of a heretofore poorly understood neuroendocrinestructure, commonly known as the vomeronasal organ (“VNO); also known as“Jacobson's organ”), with one or more steroid(s) or with compositionscontaining the steroid(s). This organ is accessed through the nostrilsof most higher animals—from snakes to humans, and has been associated,inter alia, with pheromone reception in certain species (see generallyMuller-Schwarze & Silverstein, Chemical Signals, Plenum Press, New York(1980)). The axons of the neuroepithelia of the vomeronasal organ,located supra palatinal, form the vomeronasal nerve and have directsynaptic connection to the accessory olfactory bulb and indirect inputfrom there to the cortico-medial amygdaloid basal forebrain andhypothalamic nuclei of the brain. The distal axons of terminals nerveneurons may also serve as neurochemical receptors in the VNO. Stensaas,L. J., et al., J. Steroid Biochem. and Molec. Biol. (1991) 39:553. Thisnerve has direct synaptic connection with the hypothalamus.

Johnson, A. et al. (J. Otolarynaoloav (1985) 14:71-79) report evidencefor the presence of the vomeronasal organ in most adult humans, butconclude that the organ is probably non-functional. Contravening resultswhich suggest that the VNO is a functional chemosensory receptor arereported by Stensaas, L., et al., surra; and by Moran, D. T., et al.,Garcia-Velasco, J. and M. Mondragon; MontiBloch, L. and B. Grosser allin J. Steroid Biochem. and Molec. Biol. (1991) 39.

It is apparent that it would be desirable to identify and synthesizehuman vomeropherins and pheromones and to develop pharmaceuticalcompositions and methods of use to influence hypothalamic function. Thisinvention relates to the unexpected discovery that, when nasallyadministered to human subjects, certain neurochemical ligands,particularly 19-nor-pregnane steroids, 19-nor-pregnene steroids andrelated compounds, or pharmaceutical compositions containing19-nor-pregnanes, 19-nor-pregnenes or related compounds, specificallybind to chemoreceptors of certain nasal neuroepithelial cells and thisbinding generates a series of neurophysiological responses resulting inan alteration of hypothalamic function of an individual. When properlyadministered, the effect of certain of these compounds on thehypothalamus affects the function of the autonomic nervous system and avariety of behavioral-or physiological phenomena which include, but arenot limited to the following: anxiety, premenstrual stress, fear,aggression, hunger, blood pressure, and other behavioral andphysiological functions normally regulated by the hypothalamus. See OttoAppenzeller, The Autonomic Nervous System. An Introduction of Basic andClinical Concepts (1990); Rorner, P. I. Central nervous control ofautonomic cardiovascular function, and Levy N. M. and Martin, P. J.Neural control of the heart, both in Handbook of Physiology: Section 2:Cardiovascular System—the heart, Vol. I, Washington, D.C, 1979, AmericanPhysiological society; Fishman, A. P., et al. editors, Handbook ofPhysiology: Section 3: Respiratory System. Vol. II. Control ofBreathing, Bethesda Md. 1986. American Physiological Society.

In some instances a single 19-nor-pregnane steroid, or related compound,is administered, in some instances combinations of 19-nor-pregnanesteroids and/or related compounds are administered and in some instancesone or more 19-nor-pregnane steroids are coadministered along with oneor more estrane or estrene steroids, androstane or androstene steroidsor a related compound.

BACKGROUND OF THE INVENTION

Definitions

An “affect” is a transient feeling state. Typical negative affects arefeelings of nervousness, tenseness, shame, anxiousness, irritability,anger, rage, and the like. “Moods” are longer lasting feeling statessuch as guilt, sadness, hopelessness, worthlessness, remorsefulness,misery, unhappiness and the like. “Character traits” are more permanentaspects of an individual's personality. Typical negative charactertraits are sensitivity, regretfulness, blameworthiness, stubbornness,resentfulness, bitterness, timidness, laziness and the like.

“Pregnane steroids” are aliphatic polycyclic hydrocarbons characterizedby a four-ring steroidal structure with a methylation at the 10- and13-positions and ethylation (including unsaturated groups) at the17-position. The 19-nor compounds lack a methyl or othercarbon-containing substituent on C-10 where C-19 would normally befound. A pregnene is a subset of pregnanes commonly understood to meanthat the compound has at least one double bond.

A “chemoreceptor” is a receptor molecule displayed on the surface of a“chemosensory” neuroepithelial cell which binds in a stereospecificfashion to a particular ligand or ligands. This specific bindinginitiates a signal transduction which initiates an afferent nerveimpulse. Chemoreceptors are found, inter alia, in taste buds, olfactoryepithelium and vomeronasal tissue.

“Pregnene steroids”, as the term is used herein, are aliphaticpolycyclic hydrocarbons with a four-ring steroidal structure, at leastone double bond in the A-ring, methylation at the 10-position and13-position, ethylation (including unsaturated groups) at the17-position and an oxo, hydroxyl or hydroxyl derivative such as analkoxy, ester, benzoate, cypionate, sulfate or glucuronide, at the3-position. The 19-nor compounds lack a methyl or other carbon-containersubstituent or C-10 where C-19 would normally be found. Derivativeswhich contain these structural characteristics are also referred togenerically as pregnene steroids.

The following structure shows the four-ring steroidal structure commonto pregnane and pregnene steroids. In describing the location of groupsand substituents, the following numbering system will be employed:

“Sexually dimorphic” refers to a difference in the effect of, orresponse to, a pharmaceutical agent between males and females of thesame species.

An “effective amount” of a drug is a range of quantity and/orconcentration which brings about a desired physiological and/orpsychological effect when administered to an individual in need of thedrug. In the present case, a needy individual is one with aphysiological or behavioral trait which is normally regulated by thehypothalamus and wherein it is desirable to affect the function of thehypothalamus or the trait. The effective amount of a given drug may varydepending upon the function to be affected, the desired effect, route ofadministration, and the like. For example, when the steroid inadministered as a solution applied to the facial skin of a subject aneffective concentration is from 1 microgram/ml to 100 μg/ml, preferably10 to 50 μg/ml and most preferably 20 to 30 μg/ml. When the steroid isintroduced directly into the VNo an effective amount is about 1 picogramto about 1 nanogram, more preferably about 10 picograms to about 50picograms. When the steroid is administered to the nasal passage, byointment, cream or aerosol, or the like, an effective amount is about100 pg to about 100 micrograms, preferably about 1 ng to about 10micrograms. It follows that some drugs may be effective whenadministered by some routes, but not effective when administered byother routes.

The “hypothalamus” is the portion of the diencephalon comprising theventral wall of the third ventricle below the hypothalamic sulcus andincluding structures forming the ventricle floor, including the opticchiasma, tuber cinereum, infundibulum, and mammallary bodies. Thehypothalamus regulates the autonomic nervous system and controls severalphysiological and behavioral functions such as the so-called fight andflight responses, sexual motivation, water balance, sugar and fatmetabolism, hunger, regulation of body temperature, endocrinesecretions, and others. The hypothalamus is also the source ofvasopressin which regulates blood pressure, and oxytocin which inducesparturition and milk release. All hypothalamic functions are potentiallymodulatable by the vomeropherin therapy described herein.

A “ligand”, as used herein, is a molecule which acts as a chemicalsignal by specifically binding to a receptor molecule displayed on thesurface of a receptor cell, thereby initiating a signal transductionacross the cell surface. Binding of ligands to chemosensory receptorscan be measured. Chemosensory tissue, such as vomeronasalneuroepithelium or olfactory neuroepithelium, contains a multiplicity ofneuroreceptors cells, each displaying at least one cell surfacereceptor. Many of the receptor molecules have identical ligandspecificity. Therefore, when the tissue is exposed to a ligand for whichit has specificity (for example a exposure of the VNO to a vomeropherin)a summated change in cell surface receptor potential can be measured.

As used herein, “lower alkyl” means a branched or unbranched saturatedhydrocarbon chain of 1 to 4 carbons, such as, for example, methyl,ethyl, n-propyl, i-butyl and the like. “Alkoxy” as used herein is usedin its conventional sense to mean the group —OR wherein R is alkyl asherein defined.

A “pheromone” is a substance that provides chemical means ofcommunication between members of the same species through secretion andperipheral chemoreception. In mammals pheromones are usually detected byreceptors in the vomeronasal organ of the nose. Commonly, pheromoneseffect development, reproduction and related behaviors. A “vomeropherin”is a more general term which includes pheromones and describes asubstance from any source which functions as a chemosensory messenger,binds to a specific vomeronasal neuroepithelial receptor, and induces aphysiological or behavioral effect. The physiologic effect of a“vomeropherin” is mediated through the vomeronasal organ.

A picogram (pg) is equal to 0.001 nanograms (ng). A ng is equal to 0.001micrograms (μg). A μg is equal to 0.001 mg.

The invention is directed to a group of certain 19-nor pregnanesteroids.

A subset of 19-norpregnanes within the group are believed to be novel.Syntheses are described herein for the following compounds as designatedon the chart: Chart 1 includes 19-norpregnanes to which the invention isdirected, but do not limit its scope. The synthesis diagrams that followdepict intermediate and substructure syntheses for the preparation ofthese 19-norpregnanes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 and FIG. 2 show the EVG and vomeronasal nerve dischargefrequency, respectively, of steroid E2/P4 and control, in female rats.

FIGS. 3 through 24 show the EVG, EDA, RF, CF, EMG, BT and EEG (alpha-V,alpha-T, beta-V and beta-T) data of administration of designated19-nor-steroids in the VNO women.

FIGS. 25 through 46 show the EVG, EDA, RF, CF, EMG, BT and EEG data ofadministration of designated 19-nor-steroids in the VNO of men.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide pharmaceuticalcompositions which contain human vomeropherins or pheromones and aresuitable for nasal administration in an individual.

It is also an object of this invention to provide methods of using thesecompositions to alter hypothalamic function of an individual.

It is a further object of this invention to provide methods of usingthese compositions to affect physiological and behavioral functions ofindividuals which are normally regulated by the hypothalamus.

Finally, it is an object of this invention to provide methods ofaltering hypothalamic function which have the following advantages: 1)administration directly to the chemoreceptors in the nasal passage andthe vomeronasal organ, without pills or needles—i.e., non-invasively; 2)a mode of drug action through the nervous system and not through thecirculatory system—thus brain function can be affected withoutconsideration of the bloodbrain barrier; 3) a direct means of affectingthe hypothalamus—there is only one synaptic junction between pheromonereceptors and the hypothalamus; and, 4) providing a highly specific drugeffect, thereby greatly reducing the potential for undesirableside-effects—this because sensory nerves are addressed to a specificlocation in the brain. Additional objects, advantages and novel featuresof the invention will be set forth in part in the description whichfollows, and in part will become apparent to those skilled in the artupon examination of the following, or may be learned by practice of theinvention.

Objects of this invention are achieved by providing a pharmaceuticalcomposition suitable for nasal administration in an individual. Thecomposition contains a pharmaceutically acceptable carrier and apregnane steroid with the formula:

wherein P₁ is oxo, α- or β-hydroxy, α- or β-acetoxy, α- or β-propionoxy,α- or β-lower acetoxy, α- or β-lower acyloxy, or α- or β-benzyloxy;

“a”, “b”, “c”, “d”, “e”, “f”, “g”, “h”, “i”, “j”, “m” and “n” arealternative sites for optional double bonds, and “k” may be absent orpresent with “j” to form a triple bond;

P₂ is hydroxy, hydrogen, lower alkoxy of 1 to 6 carbon atoms, or P₂ isabsent;

P₃ is oxo, hydrogen, hydroxy, lower alkoxy of 1-6 carbon atoms or halo;

P₄ is methyl or ethyl;

P₅ is hydrogen, methyl or halo;

P₆ is hydrogen or methyl.

One class of preferred steroid compositions contain steroids wherein “d”is a double bond, and optionally “b” is present as a double bond.Another preferred class has “a”, “d” and “e” present, and g or h areoptionally present. If “g” is present in this case, then “n” isoptionally present. Another preferred class has “c” present, with “f”optionally present.

The novel class of 19-nor-pregnanes are those of the above formula,excluding the following compounds in the instances where P₃ and P₆ arehydrogen; “f “, “n” , “h” and “g” are absent; P₄ is methyl and R′ and R″are not halo:

i) when P₁ is oxo, “c” is present and P₂ and P₅ are hydrogen; theneither of “j” or “i” cannot be present alone, “m” and “j” cannot bepresent together, or “i” and “j” cannot be present together, or “m”, “j”and “k” cannot be present together, and at least one of “i”, “j” and “k”must be present;

ii) when P₁ is OH, “a”, “d” and “e” are present and P₅ is hydrogen, thenany of “j”, “i”, or “m” cannot be present alone, or “j” and “k” cannotbe present together, or “i” and “j” cannot be present together, or “m”,“j” and “k” cannot be present together, and at least one of “i”, “j”,“k” and “m” must be present;

iii) when P₁ is beta-OH, “c” is present, and P₂ and P₅ are hydrogen,then “i” and “j” cannot be present together, or “m”, “j” and “k” cannotbe present together;

iv) when P₁ is -OME and “d” and “b” are present, then either of “j” and“i” cannot be present alone;

v) when P₁ is oxo and “d” is present and P₅ is hydrogen; then “i” cannotbe present alone or “j” and “k” cannot be present together.

The term lower alkyl, lower alkoxy, etc., is meant to encompass carbonchains of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.

Other objects of this invention are achieved by providing a method ofaltering hypothalamic function and/or autonomic function in anindividual. A ligand for a chemoreceptor displayed on the surface of anasal neuroepithelial cell is provided wherein the cell is a part oftissue other than olfactory epithelia; and, the ligand is administeredwithin a nasal passage of the individual such that the ligand bindsspecifically to the chemoreceptor, resulting in an alteration ofhypothalamic function of the individual.

All embodiments of this application relate to and include the functionalequivalents of the steroid structures disclosed in these embodiments andto those modified steroids which demonstrate said functionalequivalence,

In the following chart, particularly preferred 19-nor-pregnanes areshown.

19-NORPREGNANES P E 1 2 3 4 1

KNOWN KNOWN NOVEL KNOWN 2

KNOWN KNOWN KNOWN KNOWN 3

NOVEL NOVEL NOVEL NOVEL 4

NOVEL NOVEL NOVEL NOVEL 5

NOVEL NOVEL NOVEL NOVEL 6

NOVEL NOVEL NOVEL NOVEL 7

NOVEL NOVEL NOVEL NOVEL 8

KNOWN KNOWN NOVEL NOVEL 9

NOVEL NOVEL NOVEL NOVEL 10 

NOVEL NOVEL NOVEL NOVEL 11 

NOVEL NOVEL NOVEL NOVEL 12 

NOVEL NOVEL NOVEL NOVEL 13 

NOVEL KNOWN NOVEL NOVEL P E 5 6 7 8 1

KNOWN KNOWN KNOWN KNOWN 2

KNOWN NOVEL KNOWN KNOWN 3

NOVEL NOVEL KNOWN KNOWN 4

NOVEL NOVEL NOVEL NOVEL 5

NOVEL NOVEL NOVEL NOVEL 6

NOVEL NOVEL NOVEL NOVEL 7

NOVEL NOVEL NOVEL NOVEL 8

NOVEL NOVEL NOVEL NOVEL 9

NOVEL NOVEL NOVEL NOVEL 10 

NOVEL NOVEL NOVEL NOVEL 11 

NOVEL NOVEL NOVEL NOVEL 12 

NOVEL NOVEL NOVEL NOVEL 13 

KNOWN NOVEL NOVEL NOVEL

SUBSTRUCTURE SYNTHESIS: TYPE E

Frank B. Colton, Leonard N. Nysted, Byron Riegel, and Albert L. Raymond,J. Amer. Chem. Soc., 1957, 79, 1123.

Also a commercially available substructure, for example 17α-ETHYNYL-19-NORTESTOSTERONE.

This a commercially available substructure, for example ESTRONE,ETHYNYLESTRADIOL.

Pierre Crabble, U.S. Patent 3,492,318, 1970.

This is a commercially available substructure, for example6-DEHYDROESTRONE.

See Example.

See Example.

See Example.

O. I. Fedorova, O. S. Anisimova, and G. S. Grinenko, Khim. Prir.Soedin., 1976, 2, 180.

Frank B. Colton, Lenoard N. Nysted, Byron Riegel, and Albert L. Raymond,J. Amer. Chem. Soc., 1957, 79, 1123.

This is a commercially available substructure, for example EQUILIN.

This a commercially available substructure, for example EQUILENIN.

See Example.

See Example.

See Example.

SUBSTRUCTURE SYNTHESES: TYPE P

O. I .Fedorova, O. S. Anisimova, and G. S. Grinenko, Khim. Prir.Siedin., 1976, 2, 180.

Richard H. Peters, David F. Crowe, Mitchell A. Avery, Wesley K.M. Chong,and Masato Tanabe, J. Med. Chem. 1989, 32, 1642.

Also See Example.

Frank B. Colton, Leonard N. Nysted, Byron Riegel, and Albert L. Raymond,J. Amer. Chem. Soc., 1957, 79, 1123.

Richard H. Peters, David F. Crowe, Mitchell A. Avery Wesley K. M. Chong,and Masato Tanabe, J. Med. Chem., 1989, 32, 1642.

H. Kaufmann, P. Wieland, and J. Kalvoda, Helv. Chim. Acta., 1972, 55(2),381.

O. I. Fedorova, O. S. Anisimova, and G. S. Grinenko, Khim. Prir.Soedin., 1976, 2, 180.

Richard H. Peters, David F. Crowe, Mitchell A. Avery, Wesley K. M.Chong, and Masato Tanabe, J. Med. Chem., 1989, 32, 1642.

Peter Kaspar and Herbert Witzel, J. Steroid Biochem., 1985, Vol. 23, No.3, p. 259.

Richard H. Peters, David R. Crowe, Mitchell A. Avery, Wesley K.M. Chong,and Masato Tanabe, J. Med. Chem., 1989, 32, 1642.

Frank B. Colton, U.S. Pat. No. 2,840,582.

Pierre Crabble and Esperanza Velarde, U.S. Pat. No. 3,681,410, 1972.

Peter Kaspar and Herbert Witzel, J. Steroid. Biochem., 1985, Vol. 23,No. 3, P. 259.

Pierre Crabble, U.S. Pat. No. 3,492,318, 1970.

Klaus Prezewowsky and Rudolf Wiechert, U.S. Pat. No. 3,682,983, 1972.

METHYLNORPREGNANES

19-Norpregnanes in this series may be prepared with a methyl group inthe 6α, 7α, 18, 20, or 21 positions.

U.S. Pat. No. 3,681,410 teaches preparation of 6α-methyl analogs.

U.S. Pat. No. 3,682,983 teaches preparation of 18-methyl analogs.

U.S. Pat. No. 3,492,318 teaches preparation of 7α, 18, and 21-methylanalogs.

21-methyl analog:

L. A. Van Dijck, B. J. Lankwerden, J. G. C. M. Vermeer, and A. J. M.Weber, Recl. Trav. Chim. Pays-Bas Belg., 1971, 90,801.

7α, 18, 20, and 21-methyl analogs.

Richard H. Peters, David F. Crowe, Mitchell A. Averey, Wesley K. M.Chong, and Masato Tanabe, J. Med Chem., 1989, 32, 1642.

In addition certain methylated precursors are commercially available,for example:

From these, 7α-methyl or 18- methyl analogs may be made of substanceswherein estrone or 17α-ethynyl-19-nortestosterone (norethindrone) arethe precursors, repectively.

HALONORPREGNANES

U.S. Pat. No. 2,840,582 teaches the preparation of:

U.S. Pat. No. 3,681,410 teaches the preparation of:

Richard H. Peters, David F. Crowe, Mitchell A. Avery, Wesley K. M.Chong, and Masato Tanabe, J. Med. Chem., 1989, 32, 1642.

Alkoxy derivatives are prepared from their corresponding hydroxysteroids by reaction with an alkylating agent such as trimethyloxoniumfluoroborate, triethyloxonium fluoroborate or methylfluorosulfonate inan inert chlorocarbon solvent such as methylene chloride. Alternatively,alkylating agents such as alkyl halides, alkyl tosylates, alkylmesylates and dialkylsulfate may be used with a base such as NaH, KM orKOBut, silver oxide or barium oxide in polar, aprotic solvents as forexample, DMF, DMSO and hexamethylphosphoramide.

General procedures for synthetic reactions of steroids are known tothose skilled in art. Where time and temperature of reactions must bedetermined, these can be determined by a routine methodology. Afteraddition of the required reagents, the mixture is stirred under an inertatmosphere and aliquots are removed at hourly intervals. The aliquotsare analyzed by chromatography to monitor the disappearance of startingmaterial, at which point the work-up procedure is initiated. If thestarting material is not consumed within twenty-four hours, the mixtureis heated to ref lux and hourly aliquots are analyzed, as before, untilthe starting material disappears. In this case the mixture is allowed tocool before the work-up procedure is initiated.

Purification of the products is accomplished by means of chromatographyand/or crystallization, as known to those skilled in the art.

Pharmaceutical Compositions and Methods of Use

An embodiment of the subject invention is a method of altering thehypothalamic function of an individual. Another embodiment is alteringan autonomic function of an individual. These autonomic functionsinclude but are not limited to heart rate, respiratory rate, brain wavepatterns (percentage alpha cortical activity), body temperature. Otherembodiments include, but are not limited to, methods of diminishingnegative affect, negative mood or negative character traits of anindividual. Another embodiment is a method of treating femalepremenstrual stress. All of these embodiments are accomplished by meansof the non-systemic, nasal administration of certain pregnane steroids,combinations of pregnane steroids and combinations of one or morepregnane steroids and one or more androstane and/or estrene steroids.

This particular mode of administration is distinguished from alternativemodes, such as ingestion or injection, in several important ways, theseby virtue of the direct contact with the VNO provided by the nasaladministration of the steroid ligand. In the methods of this invention,the appropriate ligand is administered directly to the chemoreceptors inthe nasal passage and the vomeronasal organ, without pills orneedles—i.e., non-invasively. Drug action is mediated through binding ofthe ligands, described herein, to specific receptors displayed byneuroepithelial cells in the nose, preferably in the VNO. Thisfurthermore, the mode of drug action is through the nervous system andnot through the circulatory system—thus brain function can be affectedwithout consideration of the blood-brain barrier. These methods oftreatment provide a direct means of affecting the hypothalamus throughthe nervous system because there is only one synaptic junction betweenpheromone receptors and the hypothalamus. Because sensory nerves areaddressed to a specific location in the brain, this method has a highlyspecific drug effect, thereby greatly reducing the potential ofundesirable side-effects.

VNO contact is important because the VNO is associated withchemoreceptive/pheromonal function. The VNO consists of a pair of blindtubular diverticula which are found at the inferior margin of the nasalseptum. The VNO contains neuro-epithelia, the axons of which have directsynapses to the amygdala and from there, to the hypothalamus. Theexistence of the VNO has been well documented in most terrestrialvertebrates including the human fetus; however, in adult humans it isgenerally thought to be rudimentary (See Johnson, et al., supra).

Antifertility Activity

The steroid 19-norpregna-1,3,5(10)-trien-3-ol (compound E2/P4 in the19-norpregnane chart) was tested in the VNO of female rats. The EVG andvomeronasal nerve (VNn) discharge frequency are shown in FIGS. 1 and 2,respectively. This data shows stimulation of the VNO. The steroid E2/P4was shown to have postcoital antifertility activity when administeredorally to female rats while having low hormonal activity (measured byestrogen-receptor binding). (Peters, et al., J. Med. Chem., 1989,32,1642-52.) The data in FIGS. 1 and 2 suggest that this antifertilityactivity is explainable because E2/P4 is not a hormone, but acts as avomeropherin through stimulation of the VNO, which in turn affects thehypothalamus. Consistent with the rat model data, the compound E2/P4also shows VNO stimulation in women (see FIG. 22), and to a lesserextent, in men (see FIG. 44), and thus it is expected that thevomeropherins have antifertility activity in humans.

Stimulation of the hypothalamus via the VNO may allow one to suppressrelease of LH and FSH. This can provide a clinical method for treatmentof prostatic cancer, precocious puberty (in males and females),endometriosis, uterine leiomyoma, breast cancer, premenstrual syndromeand disfunctional uterine bleeding.

The ligand substances described herein, or their sulfated cypionated,benzoated, proprionated, or glucuronated derivatives, may beadministered directly, but are preferably administered as compositions.They are prepared in a liquid dosage form such as, for example, liquids,suspensions or the like, preferably in unit dosage forms suitable forsingle administration of precise dosages. Liquid dosages may beadministered as nose drops or as an aerosol. Alternatively, the activecompound can be prepared as a creme or an ointment composition andapplied topically within the nasal cavity. In addition, a vomeropherinmay be administered as vapor contained in an air puff delivered in thenasal cavity. As another alternative, delivery may occur by controlledrelease of these agents by encapsulation either in bulk or at amicroscopic level using synthetic polymers, such as silicone, andnatural polymers such as gelatin and cellulose. The release rate can becontrolled by proper choice of the polymeric system used to control thediffusion rate (Langer, R. S. and Peppas, N. A., Biomaterials 2,201,1981). Natural polymers, such as gelatin and cellulose slowly dissolvein a matter of minutes to hours while silicone remains intact for aperiod of months. The compositions will include a conventionalpharmaceutical carrier or excipient, one or more of the active19-nor-pregnane compound(s), and the composition may or may notadditionally include one or more androstane or estrene steroids. Inaddition, the compositions may include other medicinal agents,pharmaceutical agents, carriers, adjuvants, etc.

The most likely means of communication of a semiochemical ligand is theinhalation of a naturally occurring pheromone present on the skin ofanother. It is estimated that the naturally occurring maximumconcentration of a pregnane steroid on human skin is from 2 to 7 ng/cm².During intimate contact it is estimated that a human would be exposed tono more than 700 ng of a naturally occurring steroid. Since thesecompounds are relatively nonvolatile, it is estimated that, even duringintimate contact, a human subject would inhale no more than 0.7 pg of anaturally occurring steroid from the skin of another. From the a countinhaled only about 1% would reach the receptors of the vomeronasalorgan. Thus the estimated maximum natural exposure to naturally producedpheromones would be 0.007 pg.

The amount of vomeropherin administered will of course, be dependent onthe subject being treated, the severity of the affliction, the manner ofadministration, the frequency of administration, and the judgment of theprescribing physician. However, a single dosage of at least about 10picograms, delivered directly into the lumen of the vomeronasal organ,is effective in eliciting a transient autonomic response. Whenadministered to the nasal cavity, the dosage is about 100 picograms toabout 10 micrograms, preferably about 1 nanogram to about 10 micrograms,more preferably about 10 nanograms to 1 about microgram. The frequencyof administration is desirably in the range of an hourly dose to amonthly dose, preferably from 8 times/day to once every other day, morepreferably 1 to 3 times per day. Ointments containing one or more activecompounds and optional pharmaceutical adjuvants in a carrier, such as,for example, water, saline, aqueous dextrose, glycerol, ethanol, and thelike, can be prepared using a base such as, for example, petroleumjelly, lard, or lanolin.

Liquified pharmaceutically administrable compositions can, for example,be prepared by dissolving, dispersing, etc. an active compound asdefined above and optional pharmaceutical adjuvants in a carrier, suchas, for example, water, saline, aqueous dextrose, glycerol, ethanol, andthe like, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, for example, sodium acetate,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, etc. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art; for example, seeRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,15th Ed., 1975. The composition or formulation to be administered will,in any event, contain a quantity of one or more of the activecompound(s) in an amount effective to alleviate the symptoms of thesubject being treated.

For aerosol administration, the active ingredient is preferably suppliedin finely divided form along with a surfactant and a propellant. Typicalpercentages of active ingredients are 0.001 to 2% by weight, preferably0.004 to 0.10%.

Surfactants must, of course, be nontoxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, olestearic andoleic acids with an aliphatic polyhydric alcohol or its cyclic anhydridesuch as, for example, ethylene glycol, glycerol, erythritol, arabitol,mannitol, sorbitol, and hexitol anhydrides derived from sorbitol (thesorbitan esters sold under the trademark “Spans”) and thepolyoxyethylene and polyoxypropylene derivatives of these esters. Mixedesters, such as mixed or natural glycerides, may be employed. Thepreferred surface-active agents are the oleates or sorbitan, e.g., thosesold under the trademarks “Arlacel C” (sorbitan sesquioleate), “Span 80”(sorbitan monoleate) and “Span 85”, (sorbitan trioleate). The surfactantmay constitute 0.1-20% by weight of the composition, preferably 0.25-5%.

The balance of the composition is ordinarily propellant. Liquifiedpropellants are typically gases at ambient conditions, and are condensedunder pressure. Among suitable liquefied propellants are the loweralkanes containing up to five carbons, such as butane and propane;fluorinated or fluorochlorinated alkanes, such as are sold under thetrademark “Freon”. Mixtures of the above may also be employed.

In producing the aerosol, a container equipped with a suitable valve isfilled with the appropriate propellant, containing the finely dividedactive ingredient and surfactant. The ingredients are thus maintained atan elevated pressure until released by action of the valve.

Yet another means of administration is topical application of a volatileliquid composition to the skin, preferably facial skin, of anindividual. The composition will usually contain an alcohol such asethanol or isopropanol. A pleasant odorant may also be included in thecomposition.

Measuring Affect, Mood and Character Trait

Feeling states associated with affects, moods and character traits aregenerally measured by use of a questionnaire. For example questionnairescomprising a number of adjectives which refer to feeling states may beadministered to an individual. The individual evaluates his or herfeeling state described by the adjective and rates the intensity of thefeeling on a numerical scale. Clustering of related adjectives andstatistical analysis of a subject's evaluation of each adjectiveprovides a basis for the measurement of various feeling states.

Alternatively, feeling states may be measured by autonomic changes, suchas those used in polygraphic evaluations (galvanic skin response, pulserate and the like). Cabanac, M. Annual Review of Physiology (1975)37:415; Hardy, J. D., “Body Temperature Regulation”, Chapter 59, pp.1417. In: Medical Physiology. Vol. II Ed.: VB Mountcastle (1980);Wolfram Bouscein. Electrodermal Activity (Plenum Press 1992). Inaddition, non-verbal cues such as facial expression and body posture maybe evaluated.

EXAMPLES

The following examples are intended to illustrate but not to limit theinvention.

Abbreviations used in the examples are as follows: aq.=aqueous; RT.=roomtemperature; PE=petroleum ether (b.p. 50-70°);DMF=N,N-dimethylformamide; DMSO=dimethyl sulfoxide; THF=tetrahydrofuran.

Example 1 19-Norpregna-4,20-dien-3β-ol, 1

A suspension of 19-norpregna4,20-dien-3-one (0.38 g, 13 mmol) andlithium tri-t-butoxyaluminohydride (1.36 g, 5.35 mmol) in 20 mL of anh.ether was stirred 5 h at room temperature, following which Glauber'ssalt (6.74 g) was added. The resulting mixture was stirred 5 min. andthen filtered through a glass frit. The residue was washed with 5 20 mLportions of ether and the combined filtrates were concentrated underreduced pressure. The crude product was purified by preparative TLC onsilica gel GF using 5% ethyl acetate/methylene chloride as eluent togive a yellow resin (39.6 mg, 0.138 mmol, 11%) homogeneous to TLC (5%ethyl acetate/methylene chloride on silica gel; R_(f) 0.29).

Example 2 19-Norpregna-1,3,5(10),17,20-Pentaen-3-ol, 2

To a suspension of lithium aluminum hydride (LAH, 256.1 mg, 6.748 mmol)and aluminum chloride (296.8 mg, 2.227 mmol) in 20 mL of anh. etherunder argon was added ethinylestradiol (1.0000 g, 3.3738 mmol) in 20 mLanh. ether. After refluxing 20 h the reaction was quenched with theaddition of Glauber's salt (2.00 g) and stirring a further 2 h. Themixture was then filtered through diatomaceous earth and the residue waswashed with 3 10 mL portions of ethyl acetate. Concentration of thecombined filtrates, flash chromatography on silica gel with 15% ethylacetate/hexanes, and twofold recrystallization from aqueous ethanol gaveslightly tan needles (367.5 mg, 1.311 mmol, 39%), m.p. 132-133° C.,homogeneous to TLC (20% ethyl acetate/hexanes on silica gel; R_(f) 0.36;estra-1,3,5(10),16-tetraen-3-ol R_(f) 0.36).

Example 3 19-Norpregna-1,3,5(1)-trien-3-ol-20β-yne, 3

To a cooled (dry ice/acetone bath) solution of19-norpregna-1,3,5(10),17,20-pentaen-3-ol (2, 280.4 mg, 1.000 mmol) in28 mL of anh. THF under argon was added n-BuLi (2.5M in hexane, 1.2 mL,3.0 mmol) over 10 min. Stirring was continued for 18 h, during which thereaction was allowed to gradually warm to RT. The reaction was quenchedwith 25 mL of 1N HCl and then extracted with 3 10 portions of ether. Thecombined organic extracts were washed with 25 mL of saturated sodiumbicarbonate +25 mL of brine, dried over magnesium sulfate, and filteredthrough diatomaceous earth. The reside was washed with 10 mL of etherand the combined filtrates were concentrated under reduced pressure.Flash chromatography of the resulting yellow resin on silica gel with20% ethyl acetate/hexanes, followed by recrystallization from aqueousethanol gave fine, white needles (150.5 mg, 0.5367 mmol, 54%), m.p.148-149° C., homogeneous to TLC (20% ethyl acetate/hexanes on silicagel; R_(f) 0.34; starting material R_(f) 0.37).

Example 4 19-Norpregna-1,3,5(10),16,20-pentaen-3-ol, 4

19-Norpregna-1,3,5(10),16-tetraen-3-ol-20-yne (200.0 mg, 0.7184 mmol) in9 mL anh. THF was added to approx. 30 mL of anh. ammonia. Sodium (0.07g, 3 mg-atom) was added in small pieces and the reaction was stirred 1h, during which the color disappeared. Abs. ethanol (3 mL) was added andthe mixture was allowed to warm to RT overnight. HCl (1N, 20 mL) wasadded the mixture was extracted three times with 10 mL portions ofmethylene chloride. The combined organic extracts were washed with 10 mLof brine, dried over magnesium sulfate, and filtered throughdiatomaceous earth. The reside was washed with 10 mL of methylenechloride and the combined filtrates were concentrated under reducedpressure. Preparative TLC (silica gel GF, with 20% ethylacetate/hexanes) of the resulting amber resin, followed byrecrystallization from benzene/hexanes gave an off-white powder, m.p.123-125° C. TLC (20% ethyl acetate/hexanes) showed a major product(R_(f) 0.38) with a minor contaminant (R_(f) 0.04).

Example 5 19-Norpregna-5(10),20-dien-3-one, 5

19-Norpregna-2,5(10),20-trien-3-yl methyl ether (750.0 mg, 2.513 mmol)was dissolved in 80 mL of acetone and oxalic acid (0.88g, 7.0 mmol) in12 mL of water was added. Further acetone (20 mL) was added to bringmost of the precipitate back into solution and the reaction was stirred6 h. Following saturated sodium bicarbonate quench (30 mL) the reactionmixture was twice with 40 mL portions of ethyl acetate. The combinedorganic extracts were washed twice with 50 mL portions of brine, driedover magnesium sulfate, and filtered through diatomaceous earth. Thereside was washed with 25 mL of ethyl acetate and the combined filtrateswere concentrated under reduced pressure. Flash chromatography (10%ethyl acetate/hexanes on silica gel) gave a colorless resin (0.54 g, 1.9mmol, 76%).

Example 6 19-Norpregna-5(10),20-dien-3-ol, 6

To an ethereal (8.4 mL) solution of 19-norpregna-5(10) ,20-dien-3-one(0.42 g, 1.5 mmol) was added 69.7 mg (1.84 mmol) of LAH and the reactionwas stirred 30 min. Glauber's salt (2.79 g) was added and the suspensionwas stirred an additional 10 mL. The mixture was then filtered throughdiatomaceous earth and the residue was extracted with 435 mL portions ofether. The combined filtrates were concentrated under reduced pressureand the resulting oil was flash chromatographed (20% ethylacetate/hexanes on silica gel) to give an off-white foam (0.38 g, 1.3mmol, 88%).

Example 7 19-Norpreana-4,20-dien-10β-ol-3-one, 7

19-Norpregna-5(10),20-dien-3-one (5,0.45 g, 1.6 mmol) in DMF (5.7 mL)was cooled in an ice-acetone bath and Jones reagent (2.67M, 0.19 mL,0.51 mmol) was added. After stirring 1½ h a further 0.19 mL of Jonesreagent were added. Stirring was continued 45 min., after which 0.38 mLof Jones reagent were added. The reaction was quenched after stirring 1more hour by the addition of 2-propanol (0.38 mL). Ethyl acetate (100mL) was added and the mixture was washed with 3 50 mL portions ofwater+50 mL of brine, dried over magnesium sulfate, and filtered throughdiatomaceous earth. The residue was washed with 10 mL of ethyl acetateand the combined filtrates were concentrated under reduced pressure.Preparative TLC on alumina with 50% ethyl acetate/hexanes gave a white,crystalline film (89.2 mg, 0.297 mmol, 19%). TLC (50% ethylacetate/hexanes on silica gel) showed mostly produce (R_(f) 0.46),contaminated with a little starting material (R_(f) 0.73).

Example 8 19-Norpregna-4,9(10),20-trien-3-one, 8

A solution of 19-Norpregna-5(10),20-dien-3-one (0.34 g, 1.2 mmol) inanh. Pyridine (4.0 mL, 49 mmol) was cooled in an ice-salt bath to below−8° C. and solid pyridinium bromide perbromide (1.26 g, 3.94 mmol) wasadded at such a rate that the reaction temperature did not exceed −2° C.After stirring 1 min., 0.20 g of phenol were added, the cold bath wasremoved, and the reaction was stirre at RT for 24 h. Ethyl acetate (50mL) was added and the mixture was washed with 50 mL of 1N HCl+25 mL ofsaturated CuSO₄+25 mL of 5% sodium hydroxide+25 mL of water+25 mL ofbrine. The mixture was then dried over sodium sulfate for 4 h andafterwards filtered through a glass frit. The residue was washed with 10mL of ethyl acetate and the combined filtrates were concentrated underreduced pressure. The resulting dark syrup (512.8 mg) was taken up in 8mL of abs. ethanol, zinc dust (260.8 mg, 3.990 mg-atom) was added, andthe suspension was refluxed ½ h. The reduction mixture was filteredthrough cotton and the residue was washed with 10 mL of ethanol.Concentration of the combined filtrates and two-fold purification bypreparative TLC, first on silica gel GF (1000μ, 20% ethylacetate/hexanes as eluent) then on alumina GF (1000μ, 20% ethylacetate/hexanes), gave a nearly colorless resin (152.8 mg, 0.5410 mmol,45%) homogeneous to TLC (R_(f) 0.22, 10% ethyl acetate/hexanes on silicagel; pregna-4,20-dien-3-one R_(f) 0.25).

Example 9 19-Norpregna-1,3,5(10),20-tetraen-3-ol, 9

Ethynylestradiol diacetate (2.0004 g, 5.2576 mmol) in 100 mL of anh. THFwas added to approx. 140 mL of anh. NH3 and sodium (1.88 g, 81.8mg-atom) was added in small slivers over 5 min. After stirring the darkblue solution 1 h, abs. ethanol was added and the reaction was allowedto gradually warm to RT overnight. 100 mL of 1N HCl were added, thelayers were separated, and the aqueous layer was extracted twice with 50mL portions of ether. The combined organic phases were washed with 3 100mL portions of brine, drived over magnesium sulfate, and filteredthrough diatomaceous earth. The residue was washed with 25 mL of etherand the combined filtrates were concentrated under reduced pressure. Theresidue was taken up in 25 mL of methylene chloride, dried over sodiumsulfate, and filtered through diatomaceous earth. The residue was washedtwice with 10 mL portions of methylene chloride and the combinedfiltrates were concentrated under reduced pressure. Flash chromatography(15% ethyl acetate/hexanes on silica gel) gave a white, crystallinesolid with yellow spots (0.86 g, 3.0 mmol, 58%).

Example 10 19-Norpregna-1,3,5(10),20-tetraen-3-yl methyl ether, 10

To crude 19-Norpregna-1,3,5(10),20-tetraen-3-ol (9, 0.86g, 3.0 mmol) in75 mL of 90% ethanol was added potassium carbonate (6.73 g, 55.2 mmol)and the suspension was refluxed ½ h. Dimethyl sulfate (0.75 mL) wasadded and the reaction was refluxed a further ½ h. Dimethyl sulfate wasadded in 3 1.8 mL aliquots (total=6.15 mL, 65.0 mmol) over 1 h, andreflux was continued for ½ h. Ice water (65 mL) was added and themixture was cooled in an ice bath and stirred for 2 h. The suspensionwas centrifuged and then filtered through a coarse frit. The residue waswashed with 50 mL of water+50 mL of 5% sodium hydroxide+3 50 mL portionsof water. The residue was recrystallized from aqueous ethanol to givefine white needles, m.p. 108.5-110° C. (lit.m.p. 108-110° C.).

Example 11 19-Norpreana-1,3,5(10),16-tetraen-6-on-20-yn-3-yl acetate, 11

Chromium trioxide (2.68 g, 2.68 mmol) was suspended in 40 mL ofmethylene chloride and the suspension was cooled in an ice-salt bath to−8° C. 3,5-Dimethylpyrazole (2.58 g, 26.8 mmol) was added and thesuspension was stirred 20 min.19-Norpregna-1,3,5(10),16-tetraen-20-yn-3-yl acetate (0.86 g, 2.7 mmol)was added over 5 min., so that the reaction temperature did not exceed−7° C. After stirring an additional 2 h, the reaction mixture was pouredthrough a 30 mm×116 mm column of silica gel and elution was continuedunder pressure with methylene chloride. Concentration of appropriatefractions gave a brown film (0.16 g, 0.48 mmol, 18%).

Example 12 19-Norpregna-1,3,5(10),16-tetraene-3,6β-diol-20-yne, 12

Crude 19-nonpregna-1,3,5(10),16-tetraen-6-on-20-yn-3-yl acetate (11,0.16 g, 048 mmol) was suspended in 20 anh. ether, LAH (36.7 mg, 0967mmol) was added, and the mixture was refluxed with exclusion of waterfor 18 h. After cooling, 1.22 g of Glauber's salt were added and thesuspension was stirred ½ h. The mixture was filtered throughdiatomaceous earth and the residue was washed with 4 10 mL portions ofhot ethyl acetate. Concentration of the combined filtrates, followed bypurification by preparative TLC (50% ethyl acetate/hexanes on silica gelGF, 1000μ) gave a white solid (26.0 mg, 88.3 μmol, 18%) homogeneous toTLC (50% ethyl acetate/hexanes on silica gel; R_(f) 0.48).

Example 13 19-Norpregna-1,3,5(10),17-tetraen-3-ol, 13

Ethyltriphenylphosphonium bromice (1.3947 g, 3.7572 mmol) and potassiumt-butoxide (422.5 mg, 3.765 mmol) suspended in anh. DMSO (4.1 mL) underargon were placed in an oil bath (80-84° C.) and stirred 1 h. Equilin(200.2 mg, 0.7459 mmol) in 4.1 mL of anh. DMSO was added and thereaction was stirred a further hour. After cooling, 25 mL of ice-1 N HClwas added and the mixture was extracted three times with 20 mL portionsof ether. The combined organic extracts were washed with 25 mL ofsaturated sodium bicarbonate+25 mL of brine, dried over magnesiumsulfate, and filtered through diatomaceous earth. The residue was washedwith 10 mL of ether and the combined filtrates were concentrated underreduced pressure. Flash chromatography (20% ethyl acetate/hexanes onsilica gel) followed by preparative TLC (20% ethyl acetate/hexanes onsilica gel GF, 1000μ) gave a slightly yellow resin (182.9 mg, 0.6523mmol, 87%) homogeneous to TLC (20% ethyl acetate/hexanes R_(f) 0.42).

Example 14 19-Norpregna-1,3,5(10),617-pentaen-3-ol, 14

Ethyltriphenylphosphonium bromide (1.3945 g, 3.7561 mmol) and potassium_(t)-butoxide (422.8 mg, 3.768 mmol) suspended in 4. J mL of anh. DMSOunder argon were placed in a 77-79° C. bath and were stirred 1 h.6-Dehydroestrone (200.4 mg, 0.7466 mmol) in 4.1 mL of anh. DMSO wasadded and the reaction was stirred in 1 h. The reaction mixture wasallowed to cool and was then poured into 25 mL o fice-1N HCl. Themixture was extracted three times with 20 mL of ether and the combinedorganic extracts were washed with 25 mL of saturated sodiumbicarbonate+25 mL of brine, dried over magnesium sulfate, and filteredthrough diatomaceous earth. The residue was washed with 10 mL of etherand the combined filtrates were concentrated under reduced pressure.Flash chromatography (15% ethyl acetate/hexanes) and preparative TLC(15% ethyl acetate/hexanes on silica gel GF, 1000μ) gave an off-whitecrystalline solid (212.9 mg, >100%) homogeneous to TLC (15% ethylacetate/hexanes on silica gel, R_(f) 0.21).

Example 15 19-Norpregna-1,3,5(10),6,8,17-hexaen-3-ol, 15

Ethyltriphenylphosphonium bromide (1.3945 g, 3.7561 mmol) and potassiumt-butoxide (422.3 mg, 3.763 mmol) suspended in 4.1 mL of anh. DMSO underargon were placed in an oil bath (74-83° C.) and the reaction wasstirred 1 h. Equilenin (200.2 mg, 0.7518 mmol) in 4.1 mL of anh. DMSOwas added and the reaction mixture was stirred a further hour. Themixture was poured into 25 mL of ice-1N HCl and extracted three timeswith 20 mL portions of ether. The combined organic extracts were washedwith 25 mL of saturated sodium bicarbonate+25 mL of brine, dried overmagnesium sulfate, and filtered through diatomaceous earth. The residuewas washed with 10 mL of ether and the combined filtrates wereconcentrated under reduced pressure. Flash chromatography (20% ethylacetate/hexanes) and preparative TLC (20% ethyl acetate/hexanes onsilica gel GF, 1000μ) gave a light yellow, crystalline wax (180.6 mg,0.6487 mmol, 86%) homogeneous to TLC.

Example 16 Electrophysiological Studies

The following electrophysiological studies were performed in clinicallynormal human volunteers of both sexes whose ages ranged from 19 to 29years. No anesthetics were used, and female subjects were excluded ifpregnant.

The stimulation and recording system consists of a “multifunctionalminiprobe” described elsewhere (Monti-Bloch, L. and Grosser, B.l. (1991)“Effect of putative pheromones on the electrical activity of the humanvomeronasal organ and olfactory epithelium,” J. Steroid Biochem. Molec.Biol. 39:573582). The recording electrode is a 0.3 mm silver ballattached to a small (0.1 mm) silver wire insulated with Teflon© thesurface of the electrode is first treated to produce a silver chlorideinterface, and is then covered with gelatin It is positioned within asmall caliber Teflon© catheter (dia=5 mm) such that the tip of theelectrode protrudes approximately 2 mm. The Teflon© catheter is 10 cm inlength and constitutes the terminal extension for a multichanneldelivery system which delivers a continuous air stream carrying discreetpulses of chemosensory stimuli. The air stream first passes into a smallchamber and is bubbled through a solution containing either avomeropherin or an olfactant in a diluent or the diluent alone. Asolenoid is used to rapidly redirect the air stream from the chamber toa route which bypasses the chamber. This creates a discreet pulse ofstimulant in the air stream. A second, outer Teflon© tube with adiameter of 2 mm surrounds the catheter-electrode assemblage, and itscentral end is connected to an aspirator that provides continuoussuction of 3 ml/s. This concentric arrangement of the outer suction tubeallows the emitted chemosensory stimuli to be localized to an area wecall a “minifield” (approx. dia=1 mm), and it avoids diffusion ofsubstances either to the area outside the intended stimulation site orinto the respiratory system. The entire stimulating and recordingassemblage may be positioned either on the neurosensory epitheliumwithin the VNo, or on the surface of the olfactory or respiratoryepithelium.

Electro-vomeronasogram (EVG): Recordings are carried out in a quiet roomwith the subject supine; the multi-functional miniprobe is initiallystabilized within the nasal cavity using a nasal retractor placed in thevestibule. Reference and ground electrodes consist of silver discs (8mm), both of which are positioned on the glabella.

The entrance to the VNO, or vomeronasal pit, is identified by firstdilating the nasal aperture and vestibule. A 6×magnifying binocularloupe with halogen illumination is then used to introduce the tip of theTeflon© catheter and recording electrode assemblage into the VNo openingwhere it is stabilized at an approximate depth of 1 mm within thevomeronasal passage. Optimal placement of the recording electrode issignaled after testing for an adequate depolarization in response to atest substance.

Electrical signals from the recording electrode are fed to a DCamplifier after which they are digitized, computer monitored, andstored. The peak-to-peak amplitude of the signals is measured, and thearea under the depolarization wave is integrated, while continuouslymonitoring the signal both on the computer screen and on a digitaloscilloscope. Artifacts produced by respiratory movements are deleted bytraining the subjects to practice mouth breathing with velopharyngealclosure. Samples of vomeropherins in concentration of 25-800 fmoles aredelivered in the continuous air stream for durations from 300milliseconds to 1 second. Usually, intervals of 3 to 5 minutes separatedeach series of short test pulses. All components of the lines carryingthe test stimuli are made of Teflon©, glass or stainless steel and arecarefully cleaned and sterilized before each use. Activity was recordedusing standard electroencephalographic (EEG) electrodes placed atpositions Cz-A1 and Tz-Al of the international 10120 system; the groundelectrode was placed on the mastoid process. Skin temperature (ST) wasrecorded by a small (1.0 mm) thermistor probe placed in the right earlobe. Respiratory frequency (RF) was measured with an adjustable straingauge placed around the lower thorax. All electrical signals were DCamplified, digitized (MP-100, Biopac systems) and continuously monitoredutilizing a computer.

Statistical Analysis: EVGs, peak-to-peak changes and frequency changesof other parameters were measured and statistically analyzed. Thesignificance of the results was determined by either using pairedt-tests or analysis of variance (ANOVA).

Reflex Effects of Vomeropherins: Studies were conducted to determine thecentral nervous system (CNS) reflex responses to vomeropherinstimulation of the VNO. The sexually dimorphic local responses inducedby vomeropherins were sometimes mirrored in the autonomic response ofmale & female subjects.

Cortical activity was recorded from Cz and Tz in male and femalesubjects during application to the VNO of air pulses (300 ms to 1 sec)containing 200 fmoles of vomeropherin. There is also preliminaryevidence that the EVG is not associated with trigeminal nociceptorendings since application of a local anesthetic (2% lidocaine) to therespiratory epithelium of the nasal septum neither blocks nor diminishesthe EVG (Monti-Bloch, L. and Grosser, B.l. (1991) “Effect of putativepheromones on the electrical activity of the human vomeronasal organ andolfactory epithelium,” J. Steroid Biochem. Molec. Biol. 39:573-582.),also, subjects failed to report sensations of pain as a consequence ofany of the stimulation procedures.

The following study compares the effect of 23 vomeropherins with19-norpregnane structure, and placebo (propylene glycol), on autonomicactivity and EEG. Twelve healthy human subjects (6 women and 6 men),ages 19 to 29 participated in this study. All substances were deliveredairborne to the vomeronasal organ (VNO) puff lasting 5 seconds. For thispurpose we use a miniprobe electrode described elsewhere, that allowedlocal stimulation and simultaneous recording of the organ'selectrovomerogram (EVG). The parameters recorded were: electrodermalactivity (EDA), respiratory frequency (RF), electrocardiogram (CF),electromyogram (EMG), body temperature (BT), and EEG from CzAl and T3A1.Autonomic activity, EEG and EVG were recorded using surface electrodes.All the techniques used were non invasive. The procedure was done in tworecording sessions each lasting one hour. The electrical recordings wereamplified, dignitized, and monitored and stored in a computer.Processing and analysis of the results were done offline.

The data on the tests on the women is shown in FIGS. 3-24, and the dataon the men is shown in FIGS. 25-46.

The results were summarized in the following tables showing the overalleffect of each vomeropherin already subtracted from control. Anarbitrary score from 0 to 5 was assigned to compare the activity of thecompounds relative to each other, but virtually all of the compoundstested had some effect.

These results show that the effect of some vomeropherins on autonomicactivity and EEG is significantly different from placebo. Also shown isthat some substances do not have significantly different effects in bothgenders.

SUMMARY OF EFFECTS OF 19-NOR PREGNANE VOMEROPHERINS ON EEG AND AUTONOMICACTIVITY IN WOMEN n = 6 EVG EDA RF CF EMG BT α-CA βCA PERFORMANCE SCOREMETHOL ETHER +25 +100 −5 −5 0 −2.5 −10 −35 EDA+, RF−, CF−, β− 5 OF E1/P8E9/P2 +5 −10 −2.5 +10 +5 +2.5 +5 −35 CF+, α+, β+ 3 E4/P2 +2.5 −10 −2 +5−.5 0 −2.5 −.2.5 EDA−, EMG− 3 E7/P1 +30 +70 −5 −5 0 0 −5 −2 EDA + RF −CF− 4 ACETATE +50 +130 0 0 0 +6 0 −15 EDA+, BT+, β3‘ 4 OF E2/P8 E3/P1+20 +70 −2.5 −2.5 0 +3 −10 −30 EDA+ 2 E10/P2 +3 −10 0 +10 −5 0 +10 +10EDA−, CF+EMG−, α+ 5 E2/P7 +25 +50 +5 −5 0 +10 −15 −30 EDA+, BT+ 3 E2/P5+25 +30 0 0 0 0 −10 −40 0 0 E2/P6 +20 +50 0 −5 0 0 −15 −30 EDA+, CF− 3E1/P1 +45 +110 +5 0 0 0 −5 −10 0 0 E2/P1 +40 +95 +3 +5 0 0 0 −15 EDA+,RF+, CF+ 4 METHOL ETHER +20 +85 −2.5 −5 0 0 −6 −20 EDA+, CF− 3 OF E2/P1E13/P1 +1 −10 0 0 −5 0 0 −40 EDA−, EMG−, β− 4 E11/P1 +20 −10 0 +5 −2.5−1 0 −25 0 0 E5/P1 0 −10 0 0 0 0 0 −40 0 0 ACETATE OF +2.5 −10 −2.5 +2.5−5 −3 +10 −20 EMG−, BT−, α+, β− 5 E6/P8 E2/P8 +25 +110 0 0 0 +10 +7 −20EDA+, BT+, α+, β− 5 E2/P2 +20 +20 −2.5 −5 0 −2.5 +7 −25 CF−, BT−, α+, β−5 E2/P4 +50 +110 +2.5 0 0 0 +30 −10 EDA+, α+, β− 4 E12/P8 +35 +100 0 −50 −3 −10 −25 EDA+, CF−, BT− 4 E8/P1 +35 +105 +2.5 0 0 −1 −10 −40 0 0METHOL ETHER +12 +95 0 −5 −2.5 −2.5 +2.5 −10 EDA+, CF− 3(+) OF E1/P8E9/P2 +20 −20 0 +2.5 −2.5 −5 −7 +40 BT− 2 E4/P2 +15 −20 0 +2.5 0 −2.5−2.5 −20 0 0 E7/P1 +18 +70 0 0 0 +7 −2.5 −30 EDA+, BT+ β− 4 ACETATE +40+80 0 0 0 +2.5 −10 −20 EDA+ 2 OF E2/P8 E3/P1 +15 +50 0 +2.5 0 −5 −10 −30EDA+, BT−, β− 4 E10/P2 +20 −20 0 +2.5 −2.5 −1 0 +40 0 0 E2/P7 +30 +120 0+2.5 0 0 −10 −20 0 0 E2/P5 +10 +90 0 0 0 +10 −5 −30 EDA+, BT+, β− 4E2/P6 +12 +100 0 0 0 −2.5 0 −20 EDA+ 2 E1/P1 +35 +120 −2.5 0 0 +2.5 −2.5−20 EDA+ 2 E2/P1 +30 +120 0 0 0 −7 −10 −20 EDA+, BT−, (EEG±) 4 METHOLETHER +15 +110 −5 0 0 0 0 −20 EDA+, RF− 3 OF E2/P1 E13/P1 +20 −20 0 0−2.5 0 −5 −15 0 0 E11/P1 +18 −20 +2.5 0 0 −2.5 −5 −15 0 0 E5/P1 +10 −200 −2.5 −2.5 0 −5 −10 0 0 ACETATE OF +2.5 −20 0 −2.5 −2.5 −2.5 −5 −15 0 0E6/P8 E2/P8 +30 +100 0 0 0 0 −10 −20 0 0 E2/P2 +15 +80 0 0 0 +2.5 −5 −250 0 E2/P4 +30 +80 −2.5 0 0 −5 −10 −20 EDA+, BT−, (EEG±) 4 E12/P8 +25+120 0 0 0 +2.5 −10 −25 EDA+ 2 E8/P1 +30 +130 0 0 0 +2.5 −5 −20 EDA+2 2

What is claimed is:
 1. A 19-norpregnane selected from the groupconsisting of: 19-norpregna-4,20-dien-3-ol,19-norpregna-4,17(20)-dien-3-ol, 19-norpregna-4,16-dien-3-ol,19-norpregn-4-en-3-ol, 19-norpregn-4-en-20-yn-3-ol,19-norpregna-4,16,20-trien-3-ol,19-norpregna-1,3,5(10),6,20-pentaen-3-ol,19-norpregna-1,3,5(10),6,17(20)-pentaen-3-ol,19-norpregna-1,3,5(10),6,16-pentaen-3-ol,19-norpregna-1,3,5(10),6-tetraen-3-ol,19-norpregna-1,3,5(10),6-tetraen-20-yn-3-ol,19-norpregna-1,3,5(10),6,16,20-hexaen-3-ol,19-norpregna-1,3,5(10),6,17(20),20-hexaen-3-ol,19-norpregna-1,3,5(10),6,16-pentaen-20-yn-3-ol,19-norpregna-4,9,20-trien-3-one, 19-norpregna-4,9,17(20)-trien-3-one,19-norpregna-4,9,16-trien-3-one, 19-norpregna-4,9-dien-3-one,19-norpregna-4,9-dien-20-yn-3-one, 19-norpregna-4,9,16,20-tetraen-3-one,19-norpregna-4,9,17(20),20-tetraen-3-one,19-norpregna-4,9,16-trien-20-yn-3-one,3-hydroxy-19-norpregna-1,3,5(10),20-tetraen-6-one,3-hydroxy-19-norpregna-1,3,5(10),17(20)-tetraen-6-one,3-hydroxy-19-norpregna-1,3,5(10),16-tetraen-6-one,3hydroxy-19norpregna-1,3,5(10)-trien-6-one,3-hydroxy-19-norpregna-1,3,5(10)-trien-20-yn-6-one,3-hydroxy-19-norpregna-1,3,5(10),16,20-pentaen-6-one,3-hydroxy-19-norpregna-1,3,5(10),17(20),20-pentaen-6-one,3-hydroxy-19-norpregna-1,3,5(10),16-tetraen-20-yn-6-one,10-hydroxy-19-norpregna-4,20-dien-3-one,10-hydroxy-19-norpregna-4,17(20)-dien-3-one,10-hydroxy-19-norpregna-4,16-dien-3-one,10-hydroxy-19-norpregn-4-en-3-one,10-hydroxy-19-norpregn-4-en-20-yn-3-one,10-hydroxy-19-norpregna-4,16,20-trien-3-one,10-hydroxy-19-norpregna-4,17(20),20-trien-3-one,10-hydroxy-19-norpregna-4,16-dien-20-yn-3-one,3-methoxy-19-norpregna-2,5(10),16-triene,3-methoxy-19-norpregna-2,5(10)-diene,3-methoxy-19-norpregna-2,5(10)-dien-20-yne,3-methoxy-19-norpregna-2,5(10),16,20-tetraene,3-methoxy-19-norpregna-2,5(10),17(20),20-tetraene,3-methoxy-19-norpregna-2,5(10),16-trien-20-yne,19-norpregna-1,3,5(10),7,20-pentaen-3-ol,19-norpregna-1,3,5(10),7,17(20)-pentaen-3-ol,19-norpregna-1,3,5(10),7,16-pentaen-3-ol,19-norpregna-1,3,5(10),7,-tetraen-3-ol,19-norpregna-1,3,5(10),7,-tetraen-20-yn-3-ol,19-norpregna-1,3,5(10),7,16,20-hexaen-3-ol,19-norpregna-1,3,5(10),7,17(20),20-hexaen-3-ol,19-norpregna-1,3,5(10),7,16-pentaen-20-yn-3-ol,19-norpregna-1,3,5,7,9,20-hexaen-3-ol,19-norpregna-1,3,5,7,9,17(20)-hexaen-3-ol,19-norpregna-1,3,5,7,9,16-hexaen-3-ol,19-norpregna-1,3,5,7,9-pentaen-3-ol,19-norpregna-1,3,5,7,9-pentaen-20-yn-3-ol,19-norpregna-1,3,5,7,9,16,20-heptaen-3-ol,19-norpregna-1,3,5,7,9,17(20),20-heptaen-3-ol,19-norpregna-1,3,5,7,9,16-hexaen-20-yn-3-ol,19-norpregna-5(10),20-dien-3-ol, 19-norpregna-5(10),17(20)-dien-3-ol,19-norpregna-5(10),16-dien-3-ol, 19-norpregn-5(10)-en-3-ol,19-norpregna-5(10)-en-2-yn-3-ol, 19-norpregna-5(10),16,20-trien-3-ol,19-norpregna-5(10),17(20),20-trien-3-ol,19-norpregna-5(10),16-dien-20-yn-3-ol,19-norpregna-1,3,5(10),20-tetraene-3,6-diol,19-norpregna-1,3,5(10),17(20)-tetraene-3,6-diol,19-norpregna-1,3,5(10),16-tetraene-3,6-diol,19-norpregna-1,3,5(10)-triene-3,6-diol19-norpregna-1,3,5(10)-trien-20-yne-3,6-diol,19-norpregna-1,3,5(10),16,20-pentaene-3,6-diol,19-norpregna-1,3,5(10),17(20),20-pentaene-3,6-diol,19-norpregna-1,3,5(10),16-tetraen-20-yne-3,6-diol,19-norpregna-5(10),20-dien-3-one, 19-norpregna-5(10),16-dien-3-one,19-norpregn-5(10)-en-3-one, 19-norpregna-5(10)-16,20-trien-3-one,19-norpregna-5(10),17(20),20-trien-3-one, and19-norpregna-5(10),16-dien-20-yn-3-one.